It is now generally acknowledged that the endocardial epithelium (endothelium) of specific segments of the primary tubular heart transforms into prevalvular tissue called "cushion mesenchyme". Some 90+ plus genes have been identified with the initiation and transformation phases of cushion cells. In contrast, almost nothing is known about the differentiation-phase of cushion morphogenesis. It is often said that "cushions remodel into valve leaflets" but how this happens is uncharted territory. Yet, valvular defects are among the most relevant and serious of all congenital heart defects whose implications may extend into adult life (e.g. calcification or prolapse). The Specific Aims are four morphogenetic hypotheses which we propose as a conceptual framework for understanding how cushions become valves. AIM 1: To determine if the persisting ("post-transformed") cushion endocardium secretes signals that initiate and pattern the outgrowth of primitive cushions to form the elongated and expanded mesenchymal cores of future valve leaflets. AIM 2:To determine the pivitol role of epicardial derived cells (EPDCs) in cushion valvulogenesis. Because the failure of EPDCs to migrate into cushions results in misshapened, hyperplastic and oversized leaflets, it is hypothesized that EPDCs promote maturation/differentiation of the endocardium which inhibits further transformation of endocardial cells into additional new mesenchyme while promoting their potential to signal interstitial growth. AIM 3: To determine potential regulatory mechanisms for cushion cell differentiation into the fibrogenic tissues of mature leaflets. The specific hypothesis is that high levels of an adhesive, extracellular protein, periostin, secreted by differentiating cushion cells, promote and/or sustain differentiation into fibrous connective tissues (and away from other pathways, e.g. cartilage, bone). AIM 4: To determine the morphogenetic mechanism(s) whereby growing valvular leaflets are released from their myocardial attachment. The specific hypothesis is based on the innovative concept that "transdifferentiation" rather than apoptosis of the associated myocardial cells frees both the leaflet from the myocardium and creates the requisite supportive tension apparatus (tendinous cords and papillary muscles). A candidate molecule for mediating the cleavage process is versican.